CDDO inhibits purified Lon but not the purified 20S proteasome. (A) Human Lon (800nM monomer) or the 20S proteasome (4nM) were preincubated for 1 hour at 37°C in the presence or absence of CDDO at the indicated concentrations, after which ATP (2 mM) and the fluorogenic dipeptide, AA2-Rh110 (6μM) were added and incubated for 3 hours at 37°C before fluorescence values were measured. (B) Purified Lon (300nM monomer) was pre-incubated in the presence or absence of CDDO at the indicated concentrations for 1 hour at 37°C, after which ATP (2mM) and TFAM (60nM) were added for 1 hour at 37°C. Samples were immunoblotted for TFAM and Lon. (C-D) Cell extracts (20 μg) from HeLa ρ0 cells treated with or without CDDO-Me (1μM; C); or, with or without epoxomicin (5μM), MG262 (5μM), CDDO-Me (1μM) or DMSO vehicle for 24 hours (D). Samples were immunoblotted with antibodies recognizing TFAM, Lon, actin, or p53.
